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Efficient, selectable marker free gene targeting in soybean using novel Ochrobactrum haywardense-mediated delivery

Kumar, S.; Lui, Z.-B.; Sanyour-Doyel, N.; Landerts, B.; Worden, A.; Anand, A.; Cho, H.-J.; Bolar, J.; Harris, C.; Huang, L.; Xing, A.; Richardson, A.

2021-10-29 plant biology
10.1101/2021.10.28.466326 bioRxiv
Show abstract

We report robust selectable marker-free gene targeting (GT) system in soybean, one of the most economically important crops. A novel efficient Ochrobactrum haywardense-mediated embryonic axis transformation method was used for the delivery of CRISPR-Cas9 components and donor template to regenerate T0 plants in 6-8 weeks after transformation. This approach generated up to 3.4% targeted insertion of the donor sequence into the target locus in T0 plants, with [~] 90% mutation rate observed at the genomic target site. The GT was demonstrated in two genomic sites using two different donor DNA templates without a need of a selectable marker within the template. High-resolution Southern by Sequencing (SbS) analysis identified T1 plants with precise targeted insertion and without unintended plasmid DNA. Unlike previous low-frequency GT reports in soybean that involved particle bombardment-mediated delivery and extensive selection, the method described here is fast, efficient, reproducible, does not require selectable marker within the donor DNA, and generates non-chimeric plants with heritable GT.

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